Clathrin coated vesicles are the most intensively studied carriers of membrane traffic in eukaryotic cells and the most prominent form of traffic from the plasma membrane to endosomes (endocytosis). Clathrin-coated pits and vesicles were the first membrane-traffic system recognized and analyzed in detail, and they have become a paradigm for efforts to understand molecular mechanisms of other modes of vesicular transport. We have recently combined high spatial-resolution snapshots from cryoEM with data from high temporal-resolution light microscopy (with single-molecule sensitivity) to answer one long-standing question about the way the clathrin pathway operates: how does the uncoating ATPase, Hsc70, together with its co-chaperone, auxilin, catalyze coat disassembly? Each of the Aims of this proposal seeks to exploit the single-molecule sensitivity of in vitro, single-particle methods as used in that work, to analyze mechanisms of general significance for intracellular membrane traffic. In Aim 1, we will determine the molecular mechanism by which auxilin detects membrane scission. In Aim 2, we will use in vitro reconstitution experiments to dissect the steps of coated pit initiation and growth. In Aim 3, we will study the molecular organization of Eps15 and its partners (epsin, intersection, FCH01/2) -- a complex, network-like assembly, with multiple cross-interacting components. PUBLIC HEALTH RELEVANCE: Clathrin-mediated endocytosis is the route by which cells take up transferring, immunoglobulins, LDL, hormones, and signaling receptors. It is a pathway frequently usurped for cell entry by viruses and bacterial toxins, as well as a one that might be exploited in designing next-generation vectors for vaccines and gene therapy.